Abstract

Cancer gene mutations are important diagnostic, prognostic, and predictive biologic markers. Mutation detection in cancer tissues has been a real challenge for researchers as a cancer is often associated with multiple mutations and new mutations may occur as the disease progresses and during treatments. Essentially, all cancer tissues are a mosaic mixture of normal and abnormal cell clones. Therefore, a new technology that can target many mutations simultaneously with high sensitivity is exceedingly desirable. Targeted next generation sequencing permits deep sequencing of hundreds of mutations concurrently. We have used a novel next generation sequencing technology that merges multiplex PCR with ion semiconductor sequencing (AmpliSeq, Life Technology) to sequence a panel of clinically actionable cancer gene mutations on 12 tumor samples including 6 archived FFPE samples, 2 bone marrow samples, and 4 cell lines with known mutations, and a normal blood sample as a control. Each sample was run three times to assay for inter-run variability. The panel contains 739 mutations including clinically actionable mutations such as EGFR mutations in exons 18-21, BRAF V600E, KRAS mutations at codons 12, 13, and 61, and many more. Using this technology we successfully identified all known mutations previously detected using Pyposequencing or Sanger sequencing technologies. The most common somatic mutations were also confirmed by Competitive Allele-Specific TaqMan® PCR (castPCR, Life Technology). To determine the sensitivity of the test, we studied mutation positive FFPE samples with serial dilutions. Our results showed that the test can detect mutations at frequencies as low as 5% with 99% confidence. Analysis of mutation calls in different runs showed little inter-run variation. Other benefits of this novel technology are its fast turn around time, which can be as short as two days, and its low cost. Our experience demonstrates that this technology holds great potential clinical utility including diagnostic and therapeutic applications.